Metastatic disease remains the primary cause of death for patients with most types of cancer. Metastasis occurs through a series of interrelated steps, including survival of cancer cells in the circulation, adhesion to endothelial cells lining blood vessels, and growth of cancer cells to form metastases. Successfully interrupting any one of these steps will stop metastatic disease and potentially cure cancer. Research on metastasis has focused on primary tumors and organs with established metastases. Very little is known about cancer cells in the vascular system, an environment where mechanical forces and molecular interactions between malignant cells and vascular endothelium control the fate of circulating tumor cells. The intravascular microenvironment in metastasis is a fertile research area ripe for new therapeutic strategies to block this fatal step in cancer. What is required, however, is an efficient method for investigating the vascular microenvironment under physiologic, yet efficient and systematically adjustable conditions. To meet this critical need in cancer research, we have developed a microfluidic device to model key physical, molecular, and cellular components of the intravascular microenvironment in metastasis. We will use this device to test two central hypotheses: 1) chemokine receptor CXCR4 and the newly identified chemokine receptor CXCR7 have additive or synergistic effects to promote intravascular steps in metastasis; and 2) endothelial molecules including CXCR4 and CXCR7 control tissue-specific metastatic potential of circulating breast cancer cells. In Aim 1, we will engineer a microfluidic flow device to reproduce mechanical stresses of the vasculature and generate spatially- restricted gradients of chemoattractant molecules. In Aim 2, we will use the microfluidic flow system to investigate integrated functions of CXCR4 and CXCR7 on breast cancer cells in responding to the pro- metastatic chemokine CXCL12. Aim 3 will investigate endothelial-specific regulation of cancer cell adhesion and proliferation, exploiting our capabilities to integrate multiple types of endothelium into one flow system and then rapidly recover cells for analysis. Endothelial regulators of metastasis are particularly appealing therapeutic targets because these cells are less likely to develop drug resistance. Collectively, this research will develop innovative microfluidic flow models to study intravascular steps in metastasis under physiologic conditions, allowing us to identify breast cancer and endothelial molecules that can be targeted therapeutically to prevent metastatic disease.